The present invention relates to a composition showing enhanced antifungal activity comprising one or more antifungal agents and one or more food additives, to the use of food additives to enhance the antifungal properties of an antifungal agent and to a method of inhibiting fungal growth using a composition according to the invention.
Contamination in foodstuffs due to the presence of common food spoilage organisms is a recurring problem in the food industry. Most surprisingly we have observed an enhancement of antifungal activity when antifungal agents are combined with commonly used food additives. Furthermore, where the food additive shows antifungal activity we have observed an enhanced effect whereby the antifungal activity of the mixture is greater than would be expected. This enhancement or synergistic effect makes it possible to use a reduced amount of antifungal compound and/or food additive present in a foodstuff. This is particularly advantageous since it is highly desirable to minimise the amount of any additive present in foodstuffs both for human and animal consumption.
Accordingly in a first aspect the invention provides an antifungal composition comprising one or more antifungal agents and one or more food additives with the proviso that said composition does not contain nisin, lactoferrin and derivatives thereof, trichorzianine or lysozyme; the relative amounts of the antifungal agent(s) and the food additive(s) being such as to enhance the overall antifungal activity of the composition.
The food additive may also exhibit some antimicrobial activity especially antifungal activity when used in isolation as is observed, for example with food preservatives, and in this case the combination of the antifungal agent and the food additive will result in a synergistic effect where the antifongal activity of the mixture will be greater than that observed for each in isolation and compared to the sum of the individual results. It will be readily apparent to the man skilled in the art that in this case the antifumgal agent and the food additive present in the composition are not the same i.e. are different. The invention therefore extends to an antifungal composition comprising one or more antifungal agents and one or more food additives; the relative amounts of the antifungal agent(s) and the food additive(s) being such as to produce a synergistic effect. The invention further extends to the use of the synergistic composition in the methods of the invention hereinafter described further herein.
In a preferred embodiment the invention provides an antifungal composition comprising one or more antifungal agents and one or more food additives wherein one or more of said food additive(s) shows antimicrobial activity with the proviso that said composition does not contain nisin, lysozyme, or lactoferrin and derivatives thereof; the relative: amounts of the antifungal agent(s) and the food additive(s) being such as to produce a synergistic effect on the overall antifungal activity of the composition.
In a further preferred embodiment the invention provides a composition comprising one or more antifungal agents and one or more food additives selected from the group consisting of metal chelating agents, wetting agents, adjuvants, antioxidants, colourants, emulsifiers, stabilisers, surfactants, bleaching agents, pH control agents, flavours and flavour enhancers, sequestrants or food preservatives with the proviso that said composition does not contain nisin, lactoferrin and derivatives thereof, trichorzianine or lysozyme; the relative amounts of the antifungal agent and the food additive being such as to enhance the overall antifungal activity of the composition.
In a further aspect the invention provides the use of a food additive to enhance the antifungal properties of an antifungal agent.
As used herein the term enhance is used to denote an improvement in antifungal activity and this may be evidenced by, for example, an observed reduction in the concentration of antifungal agent required to give strong fungal growth inhibition e.g. more than 90% fungal growth inhibition. As used herein the term synergistic is used to denote an improvement in antifungal activity which can be demonstrated to be synergistic for example by application of the Colby Formula (Colby S. R. (1967) Weeds 15, 20-22) or in graphical representation in isobolograms as described by Parrish and Davidson (1993) (In: Antimicrobials in foods. Ed. By P. M. Davidson and A. L. Branen. Marcel Dekker, Inc. New York Examples of food additives include the following commonly used food additives: sorbic acid and sorbates (E200-E203), benzoic acid and benzoates (E210-E213), hydroxy-benzoates (E214-E219), sulphur dioxide and sulphites (E220-E228), biphenyl and derivatives (E230-E232), nitrites (E249-E250), nitrates (E251-E252), lactic acid (E270), lactates (E325-E327), citric acid and citrates (E330-E333), tartaric acid and tartrates (E334-E337), orthophosphoric acid and orthophosphates (E338-E341), malates (E350-E352), adipic acid (E355), succinic acid (E363), 1,4-heptonolactone (E370), nicotinic acid (E375), triammoniun citrate (E380), ammonium ferric citrate (E381), calcium disodium EDTA (E385), glycerol (E422), di-, tri- andpolyphosphates (E450a, E450b, E450c), fatty acids (E470), mono- and diglycerides of fatty acids (E471), esters of mono- and diglycerides of fatty acids (E472a-E472f), carbonates (E500, E501, E503, E504), gluconates (E576-E578), chlorine (E925), sodium hexametaphosphate, butylated hydroxyanisole (BHA)(E320), butylated hydroxytoluene (BHT)(E321), t-butyl hydroquinone (THBQ), propyl gallate, calcium heptonate, calcium phytate, diethyl ether, EDTA, disodium dihydrogen EDTA, ethyl acetate, glycerol mono-, di- and triacetates, glycine, oxystearin, propan-1,2-diol and propan-2-ol and sodium heptonate.
Particularly preferred food additives include sorbic acid and sorbates (E200-E203), benzoic acid and benzoates (E210-E213), hydroxy-benzoates (E214-E219), acetic acid and acetates (E260-E263), lactic acid (E270), lactates (E325-E327), citric acid and citrates (E330-E333), tartaric acid and tarttates (E334-E337), orthophosphoric acid and orthophosphates (E338-E341), malates (E350-E352), adipic acid (E355), succinic acid (E363), nicotinic acid (E375), calcium disodium EDTA (E385), fatty acids (E470), mono- and diglycerides of fatty acids (E471), esters of mono- and diglycerides of fatty acids (E472a-E472f), EDTA, disodium dihydrogen EDTA, ethyl acetate, glycerol mono-, di- and triacetates.
Further preferred additives include butylated hydroxy anisole, butylated hydroxytoluene and tertiary butyl hydroquinone.
We have found that when antifungal agents are used in the presence of calcium ions a significant reduction in the activity of the antiflngal agent is observed. This observation could restrict the usefulness of the technology in foods rich in calcium such as dairy products. We have now found that the problem associated with high calcium levels may be overcome by the use of chelating agents in association with the antifungal agent and most surprisingly that the presence of the chelating agent enhances the activity of the antifungal agent. Furthermore, we have found that it is possible to achieve this with levels of chelating agents, especially EDTA and salts thereof which are substantially lower than that recommended in the literature to obtain effective chelation.
Examples of particularly preferred food additives are metal chelating agents such as EDTA and its use as the disodium or calcium disodium salts; solvents and wetting agents such as organic acids e. g. lactic acid; adjuvants; and food preservatives including membrane permeabilising agents such as, for example, sorbic acid and its use as the calcium, sodium or potassium salts, benzoic acid and its use as the sodium potassium or calcium salts, antioxidants such as propyl gallate and C1-6 alkyl ethers such as butylated hydroxy anisole, butylated hydroxytoluene and tertiary butyl hydroquinone.
The use of acids and salt or ester derivatives thereof or C1-6 alkyl ethers as food additives is most particularly preferred.
Further examples of food additives may be found in xe2x80x98Handbook of Food Additivesxe2x80x99, edited by Michael and Irene Ash, Gower (1995).
Antifungal agents which are particularly preferred for use in the composition, methods and uses according to the invention are those which are obtainable from natural sources. It is also preferred that the antifungal agents are heat stable and preferably also stable to proteolytic degradation. Such stability is particularly advantageous since foodstuffs are often subjected to very high temperatures during preparation, processing and packaging. The antifungal composition of the invention therefore preferably comprises one or more heat stable antifungal agents.
As used herein the term heat stable is used to denote retention of antifungal activity after incubation at 85xc2x0 C. for 20 minutes or at 90xc2x0 C. for 10 minutes and the term stable to proteolytic degradation is used to denote lack of susceptibility to digestion by common proteases such as trypsin.
It is further preferred that the antifungal agents exert their activity by affecting fungal cell membranes such as by acting as membrane perturbing agents. In a particularly preferred combination the antifungal agent is a membrane perturbing agent which is heat stable and stable to proteolytic degradation.
In a further aspect the invention provides an antifungal composition comprising one or more antifungal agents wherein one or more of said antifungal agents is derived from a plant or seed derivative thereof and one or more food additives wherein at least one of said food additives is an acid or salt or ester derivative thereof or a C1-6 alkyl ether, the relative amounts of the antifungal agent(s) and the food additive(s) being such as to enhance the overall antifungal activity of the composition.
In a preferred embodiment of the above aspect of the invention we provide an antifungal composition wherein one or more of said food additive(s) shows antiflingal activity; the relative amounts of the antifungal agent(s) and the food additive(s) being such as to produce a synergistic effect on the overall antifungal activity of the composition.
In the above aspect the food additive is preferably selected from the group EDTA, sorbic acid, lacticacid, benzoic acid or a salt or ester derivative thereof, propyl gallate, butylated hydroxy anisole, butylated hydroxytoluene or tertiary butyl hydroquinone.
The antifungal agent is preferably derived from plants or seeds derived therefrom belonging to plant families such as Brassicaceae also known as Cruciferae, Compositae, Leguminosae; Amaranthaceae, Hiprocastanaceae, Saxifragaceae, Gramineae and Alliaceae and more preferably from the following genuses: Raphanus, Heuchera, Aesculus, Clitoria, Brassica, Briza, Sinapsis, Cnicus, Allium, Amaranthus, Impatiens, Mirabilis and Capsicum., most preferably from Raphanus, e. g. Raphanus sativus; Sinapsis, e. g. Sinapsis alba; Amaranthus, e. g. Amaranthus caudatus; Impatiens, e. g. Impatiens balsamina; Mirabilis, e. g. Mirabilis japa; Brassica e. g. Brassica napus. 
Examples of antifungal agents which are particularly preferred for use in the compositions, methods and uses according to the invention include those capable of being isolated from natural sources such as plants and the seeds thereof, such as for example the antifungal proteins described in Published International Patent Application Nos W092115691, W092/21699, W093/05153, W093/04586, W094/11511, W095/04754, W095/18229, W095/24486, W097/21814 and W097/21815 including Rs-AFPI, Rs-AFP2, Dm-AMP1, Dm-AMP2, Hs-AFP1, Ah-AMP1, Ct-AMP1, Ct-AMP2, Bn-AFP1, Bn-AFP2, Br-AFP1, Br-AFP2, Sa-AFP1, Sa-AFP2, Cb-AMP1, Cb-AMP2, Ca-AMP1, Bm-AMP1, Ace-AMP1, Ac-AMP1, Ac-AMP2, Mj-AMP1, Mj-AMP2, Ib-AMP1, Ib-AMP2, Ib-AMP3, Ib-AMP4 and peptides derived therefrom or antifungal proteins showing 85% sequence similarity, preferably greater than 90% sequence similarity, more preferably greater than 95% sequence similarity, most preferably 96%, 97%, 98% or 99% sequence similarity with any of said proteins and are most particularly isolatable from edible varieties thereof.
In a particularly preferred embodiment of the invention the antifungal composition comprises one or more of the plant and seed derived antifungal proteins or peptides listed above, said antifungal proteins being particularly advantageous owing to their heat stability and stability to proteolytic degradation.
Particularly preferred compositions according to the invention and for use in the methods and uses of the invention comprise one or more of the antiftngal proteins Rs-AFP2, Rs-AFP1, Sa-AFPs, Bn-AFPs, Mj-AMP2, Ac-AMP1, Ib-AMP1 or Ib-AMP2 or derivatives thereof or antifungal proteins showing greater than 85% sequence similarity, preferably greater than 90% sequence similarity, more preferably greater than 95% sequence similarity and one or more of EDTA or a salt thereof, lactic acid, potassium sorbate,. sodium benzoate, propyl gallate, butylated hydroxy anisole, butylated hydroxytoluene or tertiary butyl hydroquinone.
The structure, isolation and purification of Rs-AFP2, Rs-AFP1, Bn-AFPs Sa-AFPs, Ib-AMP1, Ib-AMP2, Mj-AMP2 and Ac-AMP1 are described in Published International Patent Applications Nos. W093/05153, W095/24486, W092/15691 and W092/21699 respectively, the teaching of which is incorporated herein by reference.
As used herein the term derivatives denotes inter alia, peptides derived from the full length sequence such as those described in Published International Patent Application No. WO 97/21815 and also those where residues have been altered with respect to the native sequence such as those described in Published International Patent Application No. WO 97/21814.
In the context of the present invention, two amino acid sequences with at least 85% similarity to each other have at least 85% similar (identical or conservatively replaced) amino acid residues in a like position when aligned optimally allowing for up to 3 gaps, with the proviso that in respect of the gaps a total of not more than 15 amino acid residues is affected. Likewise, two amino acid sequences with at least 90% similarity to each other have at least 90% identical or conservatively replaced amino acid residues in a like position when aligned optimally allowing for up to 3 gaps with the proviso that in respect of the gaps a total of not more than 15 amino acid residues is affected.
For the purpose of the present invention, a conservative amino acid is defined as one which does not alter the activity/function of the protein when compared with the unmodified protein. In particular, conservative replacements may be made between amino acids within the following groups:
(i) Alanine, Serine, Glycine and Threonine
(ii) Glutamic acid and Aspartic acid
(iii) Arginine and Lysine
(iv) Isoleucine, Leucine, Valine and Methionine
(v) Phenylalanine, Tyrosine and Tryptophan
Sequence similarity may. be calculated using methods well known in the art such as for example that described by Wilbur and Lipman (Proc. Natl. Acad. Sci. USA 80, 726-730 (1983)), Myers and Miller (Comput. Appl. Biosci. 4 11-17 (1988)) or Watterman and Eggert (J. Mol. Biol. (1987)197 723-28). The MegAlign Lipman-Pearson one pair method (using default parameters) which may be obtained from DNAstar Inc, 1228 Selfpark Street, Madison, Wis., 53715, USA as part of the Lasergene system may also be used.
Examples of other antifumgal agents include for example PR proteins such as chitinases, glucanases such as beta1,3 and beta1,6 glucanases, chitin-binding lectins, zeamatins, osmotins, thionins and ribosome-inactivating proteins. Examples of suitable chitinases and glucanases can be found for example, in Published European Patent Application EP 440304; of osmotin in Published International Patent Application No. WO 91/18984; of chitinase V in Published International Patent Application No. WO 95/05467; of chitin binding protein in Published International Patent Application No. WO 94/08009; beta1,6 glucanase in International Patent Application No. PCT/EP98102580.
The antifungal agents may be synthesised chemically or produced using recombinant DNA technology using methods well known in the art. Where the antifungal agent is produced using recombinant techniques in a microorganism host, the host used for the transformation and production of the desired agent will be a GRAS organism. GRAS organisms being those organisms such as, yeast, Pichia, lactic acid bacteria and certain E. coli strains which are regarded by the Regulatory Authorities as being xe2x80x98safexe2x80x99. Proteins thus produced using GRAS organisms may then be purified and added to foodstuffs in combination with food additives. The antifungal agents may also be used in the form of extracts such as seed or plant extracts where the extract contains the antifungal agent in partially purified or enriched form and the food additive is added to the extract. This is described more fully in the examples herein.
The methods and compositionsof the invention have been found to be . particularly effective against the common food-spoilage organisms Fusarium culmorum, Penicillium chrysogenum, Penicillium roquefortii, Alternaria sp., Cladosporium sp. Trichoderma harzianum, Penicillium nalgiovense, Penicillium commune, Mucor plumbeus, Aspergillus versicolor and Scopulariopsis brevicaulis. Samples of these organisms are available from for example The American Type Culture Collection (ATCC) in Rockville, Md., USA.
In a further aspect the invention provides a method of inhibiting fimgal growth in foodstuffs comprising exposing an environment in which said growth is to be inhibited to a fungicidally effective amount of an antifungal composition according to any of the above aspects of the invention.
In a yet further aspect the invention provides a method of inhibiting fungal growth in foodstuffs comprising applying to a foodstuff or to the locus of a foodstuff a fungicidally effective amount of an antifungal composition according to any of the above aspects of the invention.
The method and compositions of the invention are particularly suitable for use with a wide range of foods and beverages including fruits and jams and dairy products such as yoghurts, cheeses, cream desserts, milk shakes. The antiflngal compositions according to the invention are in a form suitable for use with foodstuffs for human and animal consumption. Other components of the composition may be chosen according to the nature of the foodstuff and to its method of consumption and this will be readily apparent to a man skilled in the art.
The environment in which it is desired to inhibit ftingal growth may be exposed to the composition comprising the antifiagal agent and the food additive in a variety of ways which will most usually be determined by the nature of the foodstuff to be protected. The foodstuff and the composition of the invention may, for example, be mixed together during the manufacturing process. Alternatively or additionally, the container in which the foodstuff is packaged may be sprayed with the composition before the foodstuff is added and/or sprayed with the composition after packing and/or filling. The composition of the invention may also be used in conjunction with coating products e.g. cheese wax.
In a further preferred embodiment the antifungal composition shows antifungal activity against one or more of Fusarium culmorum, Penicillium chrysogenum, Penicillium roquefortii, Alternaria sp., Cladbsporium sp., Trichoderma harzianum, Penicillium nalgiovense, Penicillium commune, Mucor plumbeus, Aspergillus versicolor and Scopulariopsis brevicaulis. 
The antifungal proteins Rs-AFP2, Ib-AMP1, Ib-AMP2, Mj-AMP2, Bn-AFPs, Sa-AFPs and Ac-AMP1 have been tested as purified proteins and as seed-extracts in combination with different food additives against food spoilage fungal strains. Examples of food additives which we have identified as potentiating antifungal activity when combined with the antifungal proteins are listed in Table 1 which also. includes an indication of the maximum amounts currently allowed in different foodstuffs. The ratio of antifungal agent andthe food additive will generally be such that the level of food additive in the composition according to the invention will be significantly lower than the maximum concentration allowed. For example the food additive may be present in the composition at or between a level two to a hundred fold lower, more preferably four to fifty fold lower and most. preferably four to ten fold lower than the maximum concentration allowed for a particular foodstuff.
The antifungal agent and the food additive will generally be present in the compositions according to the invention and for use in the method of the invention in a respective ratio of from 1:0.5 to 1:900000, more preferably from 1:10 to 1:10000 and most preferably from 1:5 to 1:500.